The DNA and protein sciences have made great strides over the past two decades. Researchers have accomplished the previously unthinkable by sequencing the entire genomes of several microorganisms. The genomes of several higher eukaryotes, including mammals, are nearly completely sequenced and available on a variety of databases. Potential use of the sequence information collected to date is limitless if links between genetic sequence and cell function can be established. In order to capitalize on the seemingly endless supply of sequenced genomes, researchers have developed genetic libraries that can be screened to associate a nucleic acid sequence with a protein or peptide or cellular function. In many instances, detection involves hybridizing to the unknown DNA sequence a probe specific for a desired sequence. Yet, such probes only detect sequence motifs, and peptide function cannot be accurately predicted by the mere presence of motifs. Alternatively, nucleic acid sequences are incorporated into a vector and introduced into a host cell. The gene product encoded by the nucleic acid is expressed and detected. Often, screening is accomplished in vitro (see, for example, DeGraaf et al., Gene, 128 (1), 13-17 (1993)). For instance, nucleic acids from a library are expressed and the peptides are collected and assayed. Yet, in vitro assays are not predictive of in vivo activity, and the data collected is not easily converted into information useful to, for example, the pharmaceutical industry.
Despite the construction of genetic libraries, much of the genome remains a mystery as to the function of encoded gene products. Genomics data does not take into account pre- and post-translational processing of gene products, nor does it give any indication as the amount of peptide produced or whether a peptide is active. Therefore, it would be advantageous and more relevant to study the vast array of proteins within a cell. The term “proteomics” has been used to refer to the large-scale analysis of proteins and functional genomics.
Traditionally, the tool used for proteomics research is two-dimensional polyacrylamide gels. Two-dimensional gel electrophoresis allows the separation of many proteins from a cell lysate based on charge and mass. Proteins separated in this manner can be quantitated, catalogued, and analyzed. However, two-dimensional gels are frequently not reproducible, and the identification of the proteins separated on the gel is not straightforward. In addition, only abundantly produced proteins can be detected, as proteins are difficult to amplify. In addition, some protein complexes, such as membrane protein complexes, are hard to separate. Moreover, two-dimensional gel electrophoresis is time-consuming and labor-intensive.
Like two-dimensional gel electrophoresis, yeast two-hybrid systems also are useful in protein research. Yeast two-hybrid systems are particularly useful in determining protein-protein interactions. However, the yeast two-hybrid system has been plagued with problems with false-negative and false-positive results and usually takes months to develop even preliminary results.
Similarly, phage display libraries are used to express and screen proteins for binding to a target molecule. In phage display libraries, peptides of interest are expressed in the phage coat and displayed to the environment. Phage display libraries have been used to screen proteins in vitro by association of the expressed peptide with a target ligand. However, the utility of phage display libraries to associate function with a genetic sequence in vitro is limited in that few targets have been identified, much less successfully expressed in their native conformation. Phage display libraries also have been utilized to identify peptides in vivo (see, for example, U.S. Pat. No. 5,622,699 (Ruoslahti et al.)) Yet, gene products identified by function in the context of phage may not necessarily have similar function or activity in other contexts or environments. For example, phage have limited utility in screening in vitro and in vivo for ligands that are efficiently internalized within a cell.
Protein arrays, similar to the DNA arrays commonly used in genomics research, are currently available for the study of protein interactions. Proteins are spotted on a metal chip, which can be exposed to cell lysates, plasma, or targets from pharmaceutical companies, to identify protein interactions. Yet, the fixation of proteins on a surface can cause unfolding of the protein and changes in active site conformations. In addition, the assays must take place in vitro. Thus, the results observed using a chip assay are not necessarily indicative of interactions that occur in vivo.
Accordingly, there remains a need to provide a method of screening genetic libraries. In particular, there remains a need in the art for a method of screening the products of nucleic acid sequences of a genetic library in their natural environment, e.g., intracellularly, to identify a gene product of interest. The present invention provides a rapid, reliable, low-cost method for observing gene product interactions and, advantageously, for characterizing or identifying the encoded gene product. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.